Barley is widely used for food production. Wildtype barley seed contains approximately 50 to 60% of starch, contained in its endosperm, that has approximately 25% amylose and 75% amylopectin. Amylose is a mostly linear α-(1-4) linked glucosyl chain with a few α-(1-6) linked glucan chains and has a molecular weight of 104 to 105 daltons. Amylopectin is a highly branched glucan in which α-(1-4) linked glucosyl chains with mostly 3 to 60 glucosyl units are connected by α-(1,6)-linkages, so that approximately 5-6% of the glucosyl linkages are α-(1,6)-linkages, and has a molecular weight of 105 to 106 daltons.
A suite of enzymes are involved in cereal starch biosynthesis including ADP-glucose pyrophosphorylases (EC 2.7.7.27), starch synthases (EC 2.4.1.21), starch branching enzymes (EC 2.4, 1.18) and starch debranching enzymes (EC 3.2.1.41 and 3.2.1.68). The first committed step of starch synthesis is synthesis of ADP-glucose from Glucose-1-P and ATP, catalyzed by the enzyme ADP-glucose pyrophosphorylase. The ADP-glucose is then used as substrate for the synthesis of starch by starch synthases which transfer glucose to the non-reducing end of pre-existing α-(1-4) linked glucosyl chain of starch. The branched glucan chains of starch, linked with α-(1-6) linkages, are formed by starch branching enzymes through the cleavage of a region of the α-(1-4) linkage glucan and subsequent transfer of the short glucan to a position on the α-(1-4) linkage glucan of starch. Excess α-(1-6) linked glucan chains are removed by debranching enzymes to maintain starch in a defined structure.
Ten starch synthase genes have been identified in the rice genome and are grouped into five distinct classes: granule-bound starch synthase (GBSS), starch synthase I (SSI), starch synthase II (SSII), starch synthase III (SSIII) and starch synthase IV (SSIV). There are two GBSS isoforms (GBSSI and GBSSII), one SSI isoform, three SSII isoforms (SSIIa [SSII-3], SSIIb [SSII-2], and SSIIc [SSII-1]), two SSIII isoforms (SSIIIa [SSIII-2] and SSIIIb [SSIII-1]), and two SSIV isoforms (SSIVa [SSIV-1] and SSIVb [SSIV-2]) in rice. Proteins corresponding to SSI, SSIIa and GBSSI have been detected within starch granules, whereas SSIIIa protein has been only detected in the soluble phase of amyloplastids. The precise role of these starch synthases individually and cooperatively in determining the final structure of the starch granule largely remains undefined although the potential roles of some starch synthases have been characterized in different organs and different species.
Mutants in starch synthases have been useful in determining the roles in some cereal species. GBSSI plays a crucial role in the biosynthesis of amylose, (Nelson et al., Biochem. Biophys. Res. Comm., 9: 297-300, 1962) but it may also contribute to the synthesis of the long chains of amylopectin (Maddelein et al., J. Bio. Chem., 269: 25150-25157, 1994).
A barley SSIIa mutant has been shown to have a high amylose phenotype with reduced starch content and reduced seed weight due to the reduction of starch biosynthesis. The mutant barley lines M292 and M342 which were homozygous for a null mutation in the gene encoding SSIIa were obtained following mutagenesis of grains of the barley variety ‘Himalaya’ with sodium azide. Mutant seeds were initially selected from progeny grain of the mutagenized population on the basis of a shrunken grain phenotype. The mutant lines were further characterized by their altered starch properties, reduced SSIIa protein level and activity, and genetically by the presence of a premature stop codon in the protein coding region of the gene encoding SSIIa which caused loss of the SSIIa enzyme in the endosperm (Morell et al., Plant Journal 34: 173-185, 2003) incorporated herein in its entirety by reference). The SSIIa mutant grain also had substantially reduced starch content and this was associated with a moderate reduction in yield when the barley plants were grown in the field. It was not known if the yield could be improved, or how, while still maintaining the high amylose phenotype.
There is a need for barley grain with improved agronomic performance and methods of producing same.